Selenium is an essential micronutrient in the diet of humans and other mammals and is known to have roles in preventing various forms of cancer (e.g., colon, prostate, lung and liver), heart disease and other cardiovascular and muscle disorders. In addition, it serves as an antiviral agent and plays a role in delaying the aging process, in delaying the progression of AIDS in HIV positive patients, in immune function, in mammalian development and in male reproduction. The means by which selenium exerts these many health benefits are poorly understood, but we have proposed that they are due largely to the presence of this element in selenoproteins as the amino acid, selenocysteine (Sec). Our program therefore focuses on the means by which Sec is incorporated into protein and the role of specific selenoproteins in human health and development. Since Sec tRNA is the only known tRNA that governs the expression of an entire class of proteins, the selenoproteins, we have developed several transgenic, knockout, conditional knockout and knockdown cell and mouse lines that perturb Sec tRNA synthesis which in turn perturbs selenoprotein expression as models to better understand selenium and selenoprotein metabolism and their roles in health. This past year, our program has focused on using RNAi technology to knockdown several selenoproteins and other proteins involved in Sec biosynthesis (e.g., SLA, SECp43, PSTK, SPS1 and 2) to elucidate their roles in selenium metabolism and to determine how this element is incorporated into protein. Our group further characterized the roles of SECp43 and SLA in the biosyntheisis of Sec and its incorporation into protein. In addition, we developed a novel assay for studying selenoprotein function that involved knockdown/knock-in and are using this technique to study many new parameters of selenoprotein function that could not readily be approached previously. Using loxP-Cre technology, we targeted the removal of selenoprotein expression in mouse liver and then replaced the selenoprotein population either fully or partially with a wild type or two different mutant Sec tRNA transgenes, respectively. The mutant Sec tRNA transgenes involve mutations in the tRNA that prevent the addition of a methyl group, 2'-O-methylribose (designated Um34), at position 37 which is the wobble position of the anticodon. The absence of Um34 results in the loss of expression of stress-related selenoproteins, while the housekeeping selenoproteins are virtually completely expressed. We had shown earlier through rescue of the selenoprotein population in a standard knockout mouse using wild type and mutant transgenes that Um34 was involved in synthesis of stress-related selenoproteins. However, our ability now to target a specific organ or tissue will permit us to focus on the role of selenium and selenoproteins in a given cell type. It should be further noted that this novel regulation of protein expression occurred at the level of translation and manifested a tissue-specific pattern. We are also expanding our targeted knockout of selenoprotein expression in various tissues and organs and have shown unequivocally a role of selenoproteins in development and disease prevention. One of our major focuses in this area over the last year has been on the role of selenoproteins in immune function. We targeted the removal of selenoprotein expression in T-cells and observed about a 50% reduction in the CD8 population and that mIL-13 was enriched more than 55 fold in males and 80 fold in females of knockout mice. IgG2a was increased about two-fold in targeted mice. These studies show thus far that selenoproteins in T-cells are associated with inflammatory allergic immune responses, have an essential role in the development/differentiation/maturation and function of T-cells and are indespensible factors in determining Th1 vs Th2 responses.